Interferon-α stimulates DExH-box helicase 58 to prevent hepatocyte ferroptosis

Background Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family. Methods The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58hep−/−. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS). Results Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m6A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m6A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis. Conclusions This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury. Supplementary Information The online version contains supplementary material available at 10.1186/s40779-024-00524-9.


Molecular cloning of genes
The related genes and their corresponding truncates mentioned in this study were amplified by PCR from cDNA obtained from mouse liver tissues, which were subsequently cloned into pcDNA3.1 vectors with Flag or V5-tag.The accuracy of the constructs was confirmed by sequencing.

MS analysis
Flag-DHX58 and its associated proteins were immunoprecipitated from hepatocytes transfected with an empty vector or Flag-DHX58.After Coomassie Blue staining, the DHX58 band and the bands with more intense signals at the Flag-DHX58 Lane were cut and then analyzed by reverse-phase nanospray liquid chromatography-tandem MS.MS analysis was performed by PTM BIO (Hangzhou, China) as previously described [1].

RNA-seq
Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA).As described previously, a cDNA library was constructed and subjected to high throughput sequencing [5].The mRNA levels of DDX/DHX family members were analyzed.

RNA extraction, real-time quantitative RT-PCR (qRT-PCR), and mouse identification
Total RNA was isolated from liver tissues and primary hepatocytes using the TRIzol reagent according to the instructions of the manufacturer.qRT-PCR was performed using the SYBR RT-PCR kit (RR430B, TaKaRa, Dalian, China) and a LightCycler (Roche, Switzerland) as described previously [4].The relative expression of the individual genes was normalized to that of the internal control using the 2 -ΔΔCt cycle threshold method in each sample.The primer sequences for real-time PCR and mouse identification were included in Additional file 1: Table S1.

Histology, immunohistochemistry, and immunofluorescence
Paraffin-embedded tissue sections were stained with hematoxylin and eosin (HE) to visualize liver pathology and calculate the necrotic area using Image Pro Plus software (v6.0) as previously described [6].
A pathologist blinded to the experimental groups scored the severity of liver I/R injury according to the score criteria of Suzuki, using a semi-quantitative grading scale of 0 -4; 0, no liver necrosis; 1, single cell necrosis; 2, up to 30% lobular necrosis; 3, up to 60% lobular necrosis; and 4, more than 60% lobular necrosis [7].The liver sections were stained with primary antibodies against F4/80, Ly6G, 4-HNE, or MDA for immunohistochemical staining.For immunofluorescence, TUNEL staining and ROS detection by dihydroethidium (DHE) were performed as previously described [6,8].

Liver function analysis
Liver function was determined by evaluating the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum using an automatic biochemical analyzer, the FDC-7000i (Shanghai, China) according to the instructions of the manufacturer.

RNA decay assay
Primary hepatocytes were isolated, seeded in 24-well plates overnight, and treated with actinomycin D (SelleckChem, USA) for the indicated times.Total RNA was extracted using TRIzol reagent and analyzed using qRT-PCR.truncates were constructed, and their association with V5-tagged DHX58 were determined in HHL5 cells using Co-IP.f Gpx4 mRNA level in primary hepatocytes with Ythdc2 overexpression or knockdown was examined by qRT-PCR (n = 4).g Total m 6 A modification level in primary hepatocytes with Mettl3 knockdown was examined by m 6 A RNA methylation assay (n = 3).h Gpx4 mRNA level in primary hepatocytes with Mettl3 knockdown was examined by qRT-PCR (n = 3).i m 6 A modification of Gpx4 mRNA in primary hepatocytes from Dhx58 f/f and Dhx58 hep-/-mice was examined by RIP-qRT-PCR (n = 3).j The association between DHX58 and Gpx4 mRNA in primary hepatocytes with

Fig. S1
Fig. S1 DHX58 expression is markedly decreased post I/R in the liver.a Volcano plot of the differently Fig. S9 Pretreatment with IFN-α can inhibit hepatic ferroptosis by stimulating DHX58.Wild-type (WT) and Dhx58 hep-/-mice were pretreated with IFN-α, and then undergone liver I/R, 4-HNE (a) and MDA (b) were